DnaK, DnaJ, and GrpE heat shock proteins of Escherichia coli activate site-specific DNA binding by the RepA replication initiator protein of plasmid P1 in a reaction dependent on ATP and Mg. We previously showed that GrpE is essential for in vitro RepA activation specifically at about 1 µM free Mg. In this paper, we demonstrate that GrpE lowers the requirement of DnaK ATPase for Mg, resulting in a large stimulation of ATP hydrolysis at about 1 µM Mg with and without DnaJ and RepA. In contrast to its effect on the Mg requirement, GrpE increases the ATP requirement for DnaK ATPase and dramatically lowers the affinity of DnaK for ATP in the absence of Mg. We propose that GrpE not only lowers the affinity of DnaK for nucleotide but, by increasing affinity of DnaK for Mg, also weakens the interactions of Mg with nucleotide prior to its release.

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